JP2020513799A - Nanovesicles from cupria widus bacterium and use thereof - Google Patents

Nanovesicles from cupria widus bacterium and use thereof Download PDF

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JP2020513799A
JP2020513799A JP2019548319A JP2019548319A JP2020513799A JP 2020513799 A JP2020513799 A JP 2020513799A JP 2019548319 A JP2019548319 A JP 2019548319A JP 2019548319 A JP2019548319 A JP 2019548319A JP 2020513799 A JP2020513799 A JP 2020513799A
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キム、ユン−クン
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Abstract

本発明は、クプリアウィドゥス属細菌由来の小胞及びその使用に関する。本発明者らは、正常人に比べて胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫などの悪性疾患、心筋病症、心房細動、異形狭心症などの心臓疾患、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病などの患者のサンプルで前記小胞が有意に減少され、前記小胞は、病原性小胞による炎症媒介体の分泌を有意に抑制すると同時に癌発生を抑制した。本発明によるクプリアウィドゥス属細菌由来の小胞は、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫などの悪性疾患、心筋病症、心房細動、異形狭心症などの心臓疾患、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の診断方法、及び前記疾患の予防又は治療用組成物を開発するための目的で有用である。【選択図】なしThe present invention relates to vesicles derived from a bacterium of the genus Cupriavidus and uses thereof. The present inventors compared with normal people, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, malignant diseases such as lymphoma, cardiomyopathy, atrial fibrillation, Heart vesicles such as variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression, the vesicles are significantly reduced, and the vesicles are pathogenic. It significantly suppressed the secretion of inflammatory mediators by sex vesicles and at the same time suppressed the development of cancer. Vesicles derived from cupriawidus bacterium according to the present invention, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma and other malignant diseases, cardiomyopathy, Atrial fibrillation, heart disease such as variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression, and a method for preventing or treating the disease are developed. Useful for the purpose. [Selection diagram] None

Description

本発明は、クプリアウィドゥス属細菌由来のナノ小胞及びその使用に関し、より具体的には、クプリアウィドゥス属細菌に由来するナノ小胞を用いた癌、心血管疾患、肺疾患、代謝疾患及び神経−精神疾患などの診断方法、及び前記小胞を含む前記疾患の予防、改善又は治療用組成物などに関する。   The present invention relates to a nanovesicle derived from a bacterium of the genus Cupriavidus and use thereof, and more specifically, cancer, a cardiovascular disease, a lung disease using a nanovesicle derived from a bacterium of the genus Cupriavidus, The present invention relates to a diagnostic method for metabolic diseases and neuro-psychiatric diseases, and a composition for preventing, ameliorating or treating the diseases including the vesicles.

２１世紀に入ってから、過去には伝染病と認識された急性感染性疾患の重要性が減る一方、ヒトとマイクロバイオームとの不調和により発生する免疫機能の異常を伴った慢性疾患が生活の質とヒトの寿命を決定する主要疾患となり疾病パターンが変わった。２１世紀の難治性慢性疾患として、癌、心血管疾患、慢性肺疾患、代謝疾患及び神経−精神疾患などが国民保健において大きい問題になっている。前記難治性慢性疾患は、原因因子による免疫機能の異常を伴った慢性炎症を特徴としている。   Since the beginning of the 21st century, the importance of acute infectious diseases, which have been recognized as infectious diseases in the past, has diminished, while chronic diseases accompanied by abnormal immune functions caused by incongruity between humans and the microbiome have become a part of life. It has become a major illness that determines quality and human longevity and changes disease patterns. Cancer, cardiovascular disease, chronic lung disease, metabolic disease, and neuro-psychiatric diseases have become major problems in national health as intractable chronic diseases of the 21st century. The refractory chronic disease is characterized by chronic inflammation accompanied by abnormal immune function due to causative factors.

人体に共生する微生物は１００兆に至り、ヒト細胞より１０倍多く、微生物の遺伝子数は、ヒトの遺伝子数の１００倍を越えることが知られている。微生物叢（ｍｉｃｒｏｂｉｏｔａあるいはｍｉｃｒｏｂｉｏｍｅ）は、与えられた生息地に存在する真正細菌（ｂａｃｔｅｒｉａ）、古細菌（ａｒｃｈａｅａ）、真核生物（ｅｕｋａｒｙａ）を含んだ微生物群集（ｍｉｃｒｏｂｉａｌ ｃｏｍｍｕｎｉｔｙ）を言う。   It is known that the number of microorganisms symbiotic with the human body reaches 100 trillion, which is 10 times more than that of human cells, and the number of genes of microorganisms exceeds 100 times that of humans. The term “microbiota” or “microbiome” refers to a microbial community including bacterium, archaea, and eukarya existing in a given habitat.

人体に共生する細菌及び周辺環境に存在する細菌は、他の細胞への遺伝子、低分子化合物、タンパク質などの情報を交換するためにナノメートルサイズの小胞（ｖｅｓｉｃｌｅ）を分泌する。粘膜は、２００ナノメートル（ｎｍ）サイズ以上の粒子が通過できない物理的な防御膜を形成する。粘膜に共生する細菌の場合には粘膜を通過できないが、細菌由来の小胞は、サイズが１００ナノメートル以下であるので、比較的自由に粘膜を通じて上皮細胞を通過した後に人体に吸収される。局所的に分泌された細菌由来の小胞は、粘膜の上皮細胞あるいは皮膚角質細胞を通じて吸収されて局所炎症反応を誘導するだけでなく、人体に吸収されて各臓器に分布する。そして、吸収された臓器で免疫及び炎症反応を調節する。例えば、大腸菌（Ｅｓｈｃｈｅｒｉｃｈｉａ ｃｏｌｉ）のような病原性グラム陰性菌に由来する小胞は、気道を通じて吸入されて肺気腫を誘発して慢性閉鎖性肺疾患（ＣＯＰＤ）を誘導する。腸を通じて吸収された場合には、局所的に大腸炎を起こす。血管内では、血管内皮細胞炎症反応を通じて全身的な炎症反応及び血液凝固を促進させる。また、インシュリンが作用する筋肉細胞などに吸収されてインシュリン抵抗性と糖尿病を誘発する。一方、有益な細菌に由来する小胞は、病原性小胞による免疫機能の異常を調節して疾病を調節することができる。   Bacteria symbiotic to the human body and bacteria existing in the surrounding environment secrete nanometer-sized vesicles for exchanging information such as genes, low-molecular compounds, and proteins with other cells. The mucosa forms a physical barrier that does not allow particles over 200 nanometers (nm) in size to pass through. In the case of bacteria that coexist with the mucous membrane, it cannot pass through the mucous membrane, but since the vesicles derived from bacteria have a size of 100 nanometers or less, they are relatively freely allowed to pass through epithelial cells through the mucous membrane before being absorbed by the human body. Locally secreted bacterial-derived vesicles are not only absorbed through mucosal epithelial cells or cutaneous keratinocytes to induce a local inflammatory reaction, but also absorbed by the human body and distributed to each organ. It then regulates immune and inflammatory responses in the absorbed organs. For example, vesicles derived from pathogenic Gram-negative bacteria, such as Escherichia coli, are inhaled through the respiratory tract and induce emphysema to induce chronic obstructive pulmonary disease (COPD). If absorbed through the intestine, it causes local colitis. In the blood vessel, it promotes systemic inflammatory reaction and blood coagulation through vascular endothelial cell inflammatory reaction. Insulin is also absorbed by muscle cells acting on insulin to induce insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can regulate diseases by regulating abnormalities of immune function by pathogenic vesicles.

細菌に由来する小胞などの因子に対する免疫反応は、インターロイキン（Ｉｎｔｅｒｌｅｕｋｉｎ、以下、ＩＬ）−１７サイトカインの分泌を特徴とするＴｈ１７免疫反応が発生する。これは、細菌由来の小胞に露出するとき、ＩＬ−６が分泌され、これがＴｈ１７免疫反応を誘導する。Ｔｈ１７免疫反応による炎症は、好中球浸潤を特徴とし、炎症が発生する過程で大食細胞、好中球などのような炎症細胞から分泌される腫瘍怪死因子−α（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ−ａｌｐｈａ、以下、ＴＮＦ−α）が疾病の発生に重要な役目を担当する。   An immune reaction against factors such as vesicles derived from bacteria is a Th17 immune reaction characterized by secretion of interleukin (hereinafter, IL) -17 cytokine. When exposed to vesicles of bacterial origin, it secretes IL-6, which induces a Th17 immune response. Inflammation caused by Th17 immune reaction is characterized by neutrophil infiltration, and tumor necrosis factor-alpha (tumor necrosis factor-alpha), which is secreted by inflammatory cells such as macrophages and neutrophils in the process of inflammation. Hereinafter, TNF-α) plays an important role in the occurrence of diseases.

クプリアウィドゥス属細菌（Ｃｕｐｒｉａｖｉｄｕｓ ｓｐｐ．）は、好気性グラム陰性桿菌として土壌及び臨床検体から分離される。このうちクプリアウィドゥス・メタリデュランス（Ｃｕｐｒｉａｖｉｄｕｓ ｍｅｔａｌｌｉｄｕｒａｎｓ）は、毒性重金属に抵抗する特徴があり、酸素を通じたリン酸化を通じてエネルギーを生産し、一般的に、病原性がないことが知られている。また、クプリアウィドゥス・ネカトール（Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒ）、クプリアウィドゥス・タイワンエンシス（Ｃｕｐｒｉａｖｉｄｕｓ ｔａｉｗａｎｅｎｓｉｓ）などのクプリアウィドゥス種は、マメ科植物で窒素を固定する細菌として知られている。しかし、クプリアウィドゥス属の細菌が細菌外に小胞を分泌するという事実は報告されなかった。特に、癌、心血管疾患、肺疾患、代謝疾患及び神経−精神疾患などの難治性疾患の診断及び治療に応用した事例は報告されたことがない。   Cupriavidus spp. Is isolated from soil and clinical specimens as an aerobic gram-negative bacillus. Of these, cupriavidus metallidurans is characterized by resisting toxic heavy metals, produces energy through phosphorylation through oxygen, and is generally known to be non-pathogenic. Further, cupriavidus species such as cupriavidus necator and cupriavidus taiwanensis are known to be legume-fixing bacteria. However, the fact that cupriavidus bacteria secrete vesicles outside the bacteria was not reported. In particular, no cases have been reported that have been applied to the diagnosis and treatment of intractable diseases such as cancer, cardiovascular disease, lung disease, metabolic disease and neuro-psychiatric disease.

本発明者らは、上記のような従来の問題点を解決するために鋭意研究した結果、メタゲノム分析を通じて正常人に比べて胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫などの悪性疾患、心筋病症、心房細動、異形狭心症などの心臓疾患、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病、又は鬱病患者由来のサンプルからクプリアウィドゥス属細菌由来の小胞の含量が有意に減少していることを確認した。また、クプリアウィドゥス属細菌に属するクプリアウィドゥス・メタリデュランス（Ｃ．ｍｅｔａｌｌｉｄｕｒａｎｓ）菌から小胞を分離して大食細胞に処理したとき、病原性小胞によるＩＬ−６及びＴＮＦ−αの分泌を顕著に抑制すると同時にマウスモデルにおいて抗癌効果があることを確認し、これに基づいて本発明を完成した。   The present inventors have conducted extensive studies to solve the above-mentioned conventional problems, and compared to normal people through metagenome analysis, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer. , Prostate cancer, head and neck cancer, malignant diseases such as lymphoma, cardiomyopathy, atrial fibrillation, heart disease such as variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or It was confirmed that the content of vesicles derived from the bacterium of the genus Cupriavidus was significantly decreased from the sample derived from the depressed patient. In addition, when vesicles were separated from C. metallidurans bacteria belonging to the bacterium of the genus Cupriavidus and treated into macrophages, IL-6 and TNF-α by pathogenic vesicles were obtained. It was confirmed that it has an anti-cancer effect in a mouse model at the same time that it suppresses the secretion of Escherichia coli, and based on this, the present invention was completed.

そこで、本発明は、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の診断のための情報提供方法を提供することを目的とする。   Therefore, the present invention provides gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive lung. It is an object of the present invention to provide an information providing method for diagnosing a disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

また、本発明は、クプリアウィドゥス属由来の小胞を有効成分として含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の予防、改善又は治療用組成物を提供することを他の目的とする。   The present invention also includes gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, which contains vesicles derived from cupriawidus as an active ingredient. Another object of the present invention is to provide a composition for preventing, improving or treating atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

しかし、本発明が達成しようとする技術的課題は、上記で言及した課題に制限されず、言及しなかったまた他の課題は、以下の記載から当業者に明確に理解されるべきである。   However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned above should be clearly understood by those skilled in the art from the following description.

上記のような本発明の目的を達成するために、本発明は、下記のステップを含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の診断のための情報提供方法を提供する：
（ａ）正常人及び被検者のサンプルから分離した細胞外小胞からＤＮＡを抽出するステップ；
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）を行った後、それぞれのＰＣＲ産物を収得するステップ；及び
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常人に比べてクプリアウィドゥス属細菌由来の細胞外小胞の含量が低い場合、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病に分類するステップ。 In order to achieve the above-mentioned object of the present invention, the present invention comprises the following steps: gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma. Provide a method of providing information for the diagnosis of cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression:
(A) extracting DNA from extracellular vesicles separated from normal and subject samples;
(B) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, and then collecting each PCR product; and (c) ) When the content of extracellular vesicles derived from the bacterium of the genus Cupriavidus is lower than that of a normal person through the quantitative analysis of the PCR product, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, Classifying as prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

また、本発明は、下記のステップを含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の診断方法を提供する：
（ａ）正常人及び被検者のサンプルから分離した細胞外小胞からＤＮＡを抽出するステップ；
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲを行った後、それぞれのＰＣＲ産物を収得するステップ；及び
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常人に比べてクプリアウィドゥス属細菌由来の細胞外小胞の含量が低い場合、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病に分類するステップ。 The present invention also includes gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, and dysmorphic angina, which include the following steps. , Provides a method of diagnosing chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression:
(A) extracting DNA from extracellular vesicles separated from normal and subject samples;
(B) performing PCR on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, and then obtaining each PCR product; and (c) the PCR product Gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck when the content of extracellular vesicles derived from cupriawidus bacteria is lower than in normal subjects through quantitative analysis. Classifying as cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

本発明の一具現例で、前記ステップ（ａ）でのサンプルは、大便、血液、小便又は唾液であってもよい。   In one embodiment of the present invention, the sample in step (a) may be stool, blood, urine or saliva.

本発明の他の具現例で、前記ステップ（ｂ）でのプライマー対は、配列番号１及び配列番号２のプライマーであってもよい。   In another embodiment of the present invention, the primer pair in step (b) may be the primers of SEQ ID NO: 1 and SEQ ID NO: 2.

また、本発明は、クプリアウィドゥス属細菌由来の小胞を有効成分として含む、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の予防又は治療用薬学的組成物を提供する。   Further, the present invention includes vesicles derived from the genus Cupriavidus as an active ingredient, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, Provided is a pharmaceutical composition for preventing or treating cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

また、本発明は、クプリアウィドゥス属細菌由来の小胞を有効成分として含む、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の予防又は改善用食品組成物を提供する。   Further, the present invention includes vesicles derived from the genus Cupriavidus as an active ingredient, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, Provided is a food composition for preventing or improving cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

また、本発明は、クプリアウィドゥス（Ｃｕｐｒｉａｖｉｄｕｓ）属細菌由来の小胞を有効成分として含む、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の予防又は治療用吸入剤組成物を提供する。   The present invention also includes gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, which contains vesicles derived from a bacterium of the genus Cupriavidus as an active ingredient. Provided is an inhalant composition for preventing or treating lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

また、本発明は、クプリアウィドゥス属細菌由来の小胞を有効成分として含む薬学的組成物を個体に投与するステップを含む、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の予防又は治療方法を提供する。   In addition, the present invention comprises a step of administering to an individual a pharmaceutical composition containing vesicles derived from Cupriawidus bacterium as an active ingredient, gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, A method for preventing or treating bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression provide.

また、本発明は、クプリアウィドゥス属細菌由来の小胞の胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の予防又は治療のため使用を提供する。   In addition, the present invention is a gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, vesicle derived from cupriawidus bacterium, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial thinning. Use for the prevention or treatment of dynamics, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

本発明の一具現例で、前記小胞は、平均直径が１０〜２００ｎｍであってもよい。   In one embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm.

本発明の他の具現例で、前記小胞は、クプリアウィドゥス属細菌から自然的又は人工的に分泌されるものであってもよい。   In another embodiment of the present invention, the vesicle may be naturally or artificially secreted from a bacterium belonging to the genus Cupriavidus.

本発明の他の具現例で、前記クプリアウィドゥス属細菌由来の小胞は、クプリアウィドゥス・メタリデュランス由来の小胞であってもよい。   In another embodiment of the present invention, the vesicles derived from the bacterium of the genus Cupriavidus may be vesicles derived from Cupriavidus metallidurans.

本発明者らは、腸内細菌の場合には体内に吸収されないが、細菌由来の小胞の場合には上皮細胞を通じて体内に吸収されて全身的に分布し、腎臓、肝臓、肺を通じて体外に排泄されることを確認し、患者の大便、血液、小便又は唾液などに存在する細菌由来の小胞のメタゲノム分析を通じて胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病患者の大便、血液、小便又は唾液に存在するクプリアウィドゥス属細菌由来の小胞が正常人に比べて有意に減少していることを確認した。また、クプリアウィドゥス属細菌の一種であるクプリアウィドゥス・メタリデュランスを体外で培養して小胞を分離した後、体外で炎症細胞に投与したとき、病原性小胞による炎症媒介体の分泌を有意に抑制させることを確認したところ、本発明によるクプリアウィドゥス属細菌由来の小胞は、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病に対する診断方法、及び前記疾患に対する予防、改善又は治療用組成物に有用に用いられ得ると期待される。   In the case of enterobacteria, the present inventors do not absorb it into the body, but in the case of bacterial-derived vesicles, they are absorbed into the body through epithelial cells and are distributed systemically. Confirmation of excretion and metagenomic analysis of bacterial-derived vesicles present in stool, blood, urine or saliva of patients, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate Cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysmorphic angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression in stool, blood, urine or saliva It was confirmed that the number of vesicles derived from the bacterium of the genus Cupriavidus was significantly reduced as compared with that of a normal person. In addition, when cupriawidus metallidurans, which is a type of bacterium of the genus Cupriavidus, is cultured in vitro and vesicles are separated, and then administered to inflammatory cells in vitro, the inflammatory mediators of pathogenic vesicles It was confirmed that the secretion was significantly suppressed, and the vesicles derived from the bacterium of the genus Cupriavidus according to the present invention were gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck. Cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression, and prevention, improvement or treatment for said disease It is expected that it can be usefully used in a composition for use.

図１ａは、マウスに細菌と細菌由来の小胞（ＥＶ）を口腔で投与した後、時間別に細菌と小胞の分布様相を撮影した写真であり、図１ｂは、口腔で投与した後１２時間目に、血液、腎臓、肝臓その他の臓器を摘出して細菌と小胞の体内分布様相を評価した結果である。FIG. 1a is a photograph of the distribution of bacteria and vesicles taken by mouth after administration of bacteria and vesicles (EV) derived from bacteria to the mouse, and FIG. 1b is a photograph taken 12 hours after the oral administration. This is the result of removing the blood, kidney, liver and other organs from the eye and evaluating the biodistribution pattern of bacteria and vesicles. 図２〜図２ｃは、胃癌患者及び正常人に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果であって、図２ａは大便、図２ｂは血液、図２ｃは小便を試料とした結果である。2 to 2c show the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing a metagenomic analysis of the vesicles derived from bacteria present in gastric cancer patients and normal subjects. Shows stool, FIG. 2b shows blood, and FIG. 2c shows urine. 図３ａ及び図３ｂは、大腸癌患者及び正常人に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果であって、図３ａは、大便、図３ｂは、小便を試料とした結果である。3a and 3b are the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after carrying out metagenomic analysis of vesicles derived from bacteria present in colorectal cancer patients and normal persons, and FIG. 3a is the result of using stool, and FIG. 3b is the result of using urine as a sample. 図４は、膵臓癌患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 4 shows the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing a metagenomic analysis of the vesicles derived from bacteria present in the blood of pancreatic cancer patients and normal individuals. 図５は、胆管癌患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 5 shows the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing metagenomic analysis of the vesicles derived from bacteria present in the blood of bile duct cancer patients and normal individuals. 図６ａ及び図６ｂは、乳癌患者及び正常人に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果であって、図６ａは血液、図６ｂは小便を試料とした結果である。6a and 6b show the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing metagenomic analysis of vesicles derived from bacteria present in breast cancer patients and normal subjects. Is blood, and FIG. 6b is the result of urine as a sample. 図７ａ及び図７ｂは、卵巣癌患者及び正常人に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果であって、図７ａは血液、図７ｂは小便を試料とした結果である。7a and 7b are the results of comparing the distribution of the vesicles derived from the bacterium of the genus Cupriavidus after performing a metagenomic analysis of the vesicles derived from the bacteria present in ovarian cancer patients and normal subjects. 7a is the result of blood, and FIG. 7b is the result of urine as a sample. 図８ａ及び図８ｂは、膀胱癌患者及び正常人に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果であって、図８ａは血液、図８ｂは小便を試料とした結果である。8a and 8b are the results of comparing the distribution of the vesicles derived from the bacterium of the genus Cupriavidus after performing a metagenomic analysis of the vesicles derived from the bacteria present in bladder cancer patients and normal subjects. 8a is the result of blood, and FIG. 8b is the result of urine as a sample. 図９は、前立腺癌患者及び正常人の小便に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 9 shows the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing a metagenomic analysis of vesicles derived from bacteria present in urine of prostate cancer patients and normal subjects. 図１０は、頭頸部癌患者及び正常人の唾液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 10 is a result of comparing the distribution of vesicles derived from a bacterium belonging to the genus Cupriavidus after performing metagenomic analysis of bacteria-derived vesicles present in saliva of head and neck cancer patients and normal persons. 図１１は、リンパ腫患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 11 shows the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing a metagenomic analysis of the vesicles derived from bacteria present in the blood of lymphoma patients and normal individuals. 図１２ａ〜図１２ｃは、心臓疾患患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果であって、図１２ａは心筋病症、図１２ｂは心房細動、図１２ｃは異形狭心症の場合を示す。12a to 12c show the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after carrying out metagenomic analysis of the vesicles derived from bacteria present in the blood of heart disease patients and normal persons. 12a shows a case of cardiomyopathy, FIG. 12b shows a case of atrial fibrillation, and FIG. 12c shows a case of variant angina. 図１３は、脳卒中患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 13 shows the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing metagenomic analysis of the vesicles derived from bacteria present in the blood of stroke patients and normal individuals. 図１４は、慢性閉塞性肺疾患（ＣＯＰＤ）患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 14 shows the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriawidus after performing a metagenomic analysis of the vesicles derived from the bacteria present in the blood of patients with chronic obstructive pulmonary disease (COPD) and normals. Is. 図１５ａ〜図１５ｃは、糖尿病患者及び正常人に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果であって、図１５ａは血液、図１５ｂは小便、図１５ｃは唾液を試料とした結果である。15a to 15c show the results of comparison of the distribution of vesicles derived from the bacterium of the genus Cupriavidus after performing a metagenomic analysis of vesicles derived from bacteria present in diabetic patients and normal persons. Is blood, FIG. 15b is urine, and FIG. 15c is saliva. 図１６は、腎不全患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 16 is a result of comparing the distribution of vesicles derived from a bacterium belonging to the genus Cupriavidus after performing a metagenomic analysis of vesicles derived from bacteria present in blood of patients with renal failure and normal subjects. 図１７は、認知症患者及び正常人の血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 17 shows the results of comparing the distribution of vesicles derived from the bacterium of the genus Cupriavidus after carrying out metagenomic analysis of the vesicles derived from bacteria present in the blood of dementia patients and normal persons. 図１８は、パーキンソン病患者及び正常人の小便に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 18 shows the results of comparison of the distribution of vesicles derived from bacteria of the genus Cupriavidus after carrying out metagenomic analysis of vesicles derived from bacteria present in urine of Parkinson's disease patients and normal subjects. 図１９は、鬱病患者及び正常人の小便に存在する細菌由来の小胞のメタゲノム分析を実施した後、クプリアウィドゥス属細菌由来の小胞の分布を比較した結果である。FIG. 19 is a result of comparing the distribution of vesicles derived from a bacterium belonging to the genus Cupriavidus after performing a metagenomic analysis of vesicles derived from bacteria present in urine of depressed patients and normal subjects. 図２０は、クプリアウィドゥス・メタリデュランス由来の小胞の細胞死滅効果を評価するために、前記小胞を大食細胞（Ｒａｗ２６４．７）に投与して細胞死滅程度を評価した結果である。FIG. 20 shows the results of evaluating the degree of cell death by administering the vesicles to macrophages (Raw 264.7) in order to evaluate the cell-killing effect of vesicles derived from Cupriavidus metallidurans. .. 図２１は、クプリアウィドゥス・メタリデュランス由来の小胞の抗炎症及び免疫調節効果を評価するために、病原性小胞である大腸菌小胞（Ｅ．ｃｏｌｉ ＥＶ）処理前にクプリアウィドゥス属細菌由来の小胞を前処理して、大腸菌小胞による炎症媒介体であるＩＬ−６及びＴＮＦ−αの分泌に及ぶ影響を評価した結果である。FIG. 21 shows cupriawidus before treatment with E. coli EV, which is a pathogenic vesicle, in order to evaluate the anti-inflammatory and immunomodulatory effects of vesicles derived from Cupriawidus metallidurans. It is a result of pre-treating vesicles derived from a genus bacterium and evaluating the effect of the Escherichia coli vesicles on secretion of IL-6 and TNF-α which are inflammation mediators. 図２２は、クプリアウィドゥス・メタリデュランス由来の小胞の抗癌効能を評価するために、クプリアウィドゥス・メタリデュランス由来の小胞をマウスに投与したプロトコルである。FIG. 22 shows a protocol in which vesicles derived from Cupriavidus metalliduran were administered to mice in order to evaluate the anticancer efficacy of vesicles derived from Cupriavidus metalliduran. 図２３は、クプリアウィドゥス・メタリデュランス由来の小胞の抗癌効能を評価するために、クプリアウィドゥス・メタリデュランス小胞を腹腔（ＩＰ）又は経口（ＰＯ）で投与して、癌細胞による腫瘍発生に及ぶ影響を評価した結果である。FIG. 23 shows that, in order to evaluate the anti-cancer efficacy of vesicles derived from Cupriawidus metallidurans, cancer was administered by intraperitoneal (IP) or oral (PO) administration of cupriawidus metallidurans vesicles. It is the result of evaluating the effect of cells on tumor development.

本発明は、クプリアウィドゥス属細菌由来の小胞及びその使用に関する。   The present invention relates to vesicles derived from a bacterium of the genus Cupriavidus and uses thereof.

本発明者らは、メタゲノム分析を通じてクプリアウィドゥス属細菌由来の小胞が正常人に比べて癌、心血管疾患、肺疾患、代謝疾患及び神経−精神疾患患者の臨床サンプルで有意に減少していることを確認して、前記疾病を診断することができることを確認した。また、クプリアウィドゥス・メタリデュランス（Ｃ．ｍｅｔａｌｌｉｄｕｒａｎｓ）菌株から小胞を最初に分離して特性を分析した結果、前記菌株由来の小胞が病原性小胞による免疫機能の異常と炎症及び癌を調節することができることを確認した。   The present inventors have found that through metagenomic analysis, vesicles derived from cupriawidus bacteria are significantly reduced in clinical samples of patients with cancer, cardiovascular disease, lung disease, metabolic disease and neuro-psychiatric disease as compared to normal people. It was confirmed that the above-mentioned diseases can be diagnosed. In addition, the vesicles were first isolated from the C. metallidurans strain and the characteristics were analyzed. As a result, the vesicles derived from the strain were found to be abnormal in immune function due to pathogenic vesicles, inflammation and cancer. It was confirmed that can be adjusted.

さて、本発明は、下記のステップを含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の診断のための情報提供方法を提供する：
（ａ）正常人及び被検者のサンプルから分離した細胞外小胞からＤＮＡを抽出するステップ；
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）を行った後、それぞれのＰＣＲ産物を収得するステップ；及び
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常人に比べてクプリアウィドゥス属細菌由来の細胞外小胞の含量が低い場合、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病に分類するステップ。 Now, the present invention includes the following steps including gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, and dysmorphic angina. , Provides informational methods for the diagnosis of chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression:
(A) extracting DNA from extracellular vesicles separated from normal and subject samples;
(B) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, and then collecting each PCR product; and (c) ) When the content of extracellular vesicles derived from the bacterium of the genus Cupriavidus is lower than that of a normal person through the quantitative analysis of the PCR product, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, Classifying as prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

本発明で用いられる用語「診断」とは、広い意味では患者の病気の実態を全ての面にわたって判断することを意味する。判断の内容は、病名、病因、病型、軽重、病状の詳細な様態、合併症の有無及び予後などである。本発明で「診断」は、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び／又は鬱病の発病有無及び疾患の程度などを判断することである。   The term “diagnosis” used in the present invention means, in a broad sense, determining the actual condition of a disease of a patient in all aspects. The contents of the judgment include the name of the disease, the etiology, the type of disease, the severity, the detailed state of the condition, the presence or absence of complications, and the prognosis. In the present invention, "diagnosis" means gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, atypical angina, chronic occlusion. It is to determine the presence or absence of onset of pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and / or depression and the degree of disease.

本発明において、前記サンプルは、大便、血液、小便又は唾液であってもよいが、これに制限されるものではない。   In the present invention, the sample may be stool, blood, urine or saliva, but is not limited thereto.

本発明で用いられる用語「メタゲノム」とは、「群遺伝体」とも言い、土壌、動物の腸など孤立した地域内の全てのウイルス、細菌、カビなどを含む遺伝体の総合を意味するもので、主に培養されない微生物を分析するために配列分析器を用いて一度に多くの微生物を同定することを説明する遺伝体の概念として用いられる。特に、「メタゲノム」は、一種のゲノム、遺伝体を言うのではなく、一つの環境単位の全ての種の遺伝体として一種の混合遺伝体を言う。これは、オミックス的に生物学が発展する過程で一つの種を定義するとき、機能的に既存の一種だけではなく、多様な種が互いに相互作用して完全な種を作るという観点から生じた用語である。技術的には、迅速配列分析法を用いて種に関係なく全てのＤＮＡ、ＲＮＡを分析し、一つの環境内での全ての種を同定し、相互作用、代謝作用を解明する技法の対象である。   The term "metagenomics" used in the present invention is also referred to as "group genetics", which means a comprehensive set of genetics including all viruses, bacteria, molds, etc. in an isolated area such as soil and animal intestine. , Mainly used as the concept of a genetic body that describes identifying many microorganisms at once using a sequence analyzer to analyze uncultured microorganisms. In particular, "metagenomics" does not refer to a type of genome or genetic body, but refers to a type of mixed genetic body as a genetic body of all species in one environmental unit. This has arisen from the perspective that when defining a single species in the course of omics biology development, not only functionally existing species but also various species interact with each other to form a complete species. Is a term. Technically, it is the object of the technique to analyze all DNA and RNA regardless of species using rapid sequence analysis method, identify all species in one environment, and to elucidate the interaction and metabolism. is there.

本発明の他の様態として、本発明は、クプリアウィドゥス属細菌由来の小胞を有効成分として含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の予防、治療又は改善用組成物を提供する。前記組成物は、食品組成物、吸入剤組成物及び薬学的組成物を含み、本発明で食品組成物は、健康機能食品組成物を含む。また、本発明の組成物は、口腔噴霧剤、鼻腔噴霧剤又は吸入剤の剤型であってもよい。   As another aspect of the present invention, the present invention is a gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck containing vesicles derived from cupriawidus bacterium as an active ingredient. Disclosed is a composition for the prevention, treatment or amelioration of cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression. The composition includes a food composition, an inhalant composition and a pharmaceutical composition, and in the present invention, the food composition includes a health functional food composition. Further, the composition of the present invention may be in the form of oral spray, nasal spray or inhalant.

本発明で用いられる用語「予防」とは、本発明による組成物の投与によって前記疾患を抑制するか発病を遅延させる全ての行為を意味する。   The term “prevention” as used in the present invention refers to any act of suppressing the above-mentioned disease or delaying the onset of the disease by administration of the composition according to the present invention.

本発明で用いられる用語「治療」とは、本発明による組成物の投与によって前記疾患に対する症状が好転するか利するように変更されるすべての行為を意味する。
本発明で用いられる用語「改善」とは、治療される状態と関連したパラメータ、例えば、症状の程度を少なくとも減少させる全ての行為を意味する。 The term "treatment" as used in the present invention means any act in which the administration of the composition according to the present invention modifies or modifies the symptoms for said disease.
The term “ameliorating” as used in the present invention means any act that at least reduces the parameters associated with the condition being treated, eg the degree of symptoms.

本発明で用いられる用語「ナノ小胞（Ｎａｎｏｖｅｓｉｃｌｅ）」あるいは「小胞（Ｖｅｓｉｃｌｅ）」とは、多様な細菌から分泌されるナノサイズの膜になった構造物を意味する。グラム陰性菌（ｇｒａｍ−ｎｅｇａｔｉｖｅ ｂａｃｔｅｒｉａ）由来の小胞、又は外膜小胞（ｏｕｔｅｒ ｍｅｍｂｒａｎｅ ｖｅｓｉｃｌｅｓ、ＯＭＶｓ）は、リポ多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ）だけでなく、タンパク質、低分子化合物及び細菌ＤＮＡとＲＮＡも有しており、グラム陽性菌（ｇｒａｍ−ｐｏｓｉｔｉｖｅ ｂａｃｔｅｒｉａ）由来の小胞は、タンパク質、低分子化合物、核酸外にも細菌の細胞壁の構成成分であるペプチドグリカン（ｐｅｐｔｉｄｏｇｌｙｃａｎ）とリポタイコ酸（ｌｉｐｏｔｅｉｃｈｏｉｃ ａｃｉｄ）も有している。本発明において、ナノ小胞あるいは小胞は、クプリアウィドゥス属細菌から自然的に分泌されるか又は人工的に生産するものであって、１０〜２００ｎｍの平均直径を有している。   The term “Nanovesicle” or “Vesicle” as used in the present invention means a nano-sized membrane structure secreted from various bacteria. Vesicles derived from gram-negative bacteria (outer membrane vesicles, OMVs) have not only lipopolysaccharides, but also proteins, low molecular weight compounds, and bacterial DNA and RNA. The vesicles derived from Gram-positive bacteria also have proteins, low molecular weight compounds, and in addition to nucleic acids, peptidoglycan (lipotidechoic acid) and peptidoglycan that are components of the cell wall of bacteria. ing. In the present invention, the nanovesicles or vesicles are naturally secreted or artificially produced from cupriavidus bacteria and have an average diameter of 10 to 200 nm.

前記小胞は、クプリアウィドゥス属細菌を含む培養液を遠心分離、超高速遠心分離、高圧処理、押出、超音波分解、細胞溶解、均質化、冷凍−解凍、電気穿孔、機械的分解、化学物質処理、フィルタによる濾過、ゲル濾過クロマトグラフィー、フリーフロー電気泳動及び毛細管電気泳動からなる群より選択された一つ以上の方法を用いて分離することができる。また、不純物の除去のための洗浄、収得された小胞の濃縮などの過程を追加で含むことができる。   The vesicles are centrifuged, culture medium containing cupriawidus bacteria, ultra high speed centrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thawing, electroporation, mechanical degradation, Separation can be accomplished using one or more methods selected from the group consisting of chemical treatment, filter filtration, gel filtration chromatography, free flow electrophoresis and capillary electrophoresis. In addition, steps such as washing for removing impurities and concentration of collected vesicles can be additionally included.

本発明の一実施例では、細菌及び細菌由来の小胞をマウスに経口投与して細菌及び小胞の体内吸収、分布及び排泄様相を観察した結果、細菌の場合には、腸粘膜を通じて吸収されないのに比べて、細菌由来の小胞は、投与５分以内に吸収されて全身的に分布し、腎臓、肝臓などを通じて排泄されることを確認した（実施例１参照）。   In one embodiment of the present invention, bacteria and vesicles derived from bacteria were orally administered to mice, and as a result of observation of in vivo absorption, distribution and excretion of bacteria and vesicles, it was found that bacteria were not absorbed through the intestinal mucosa. In contrast, it was confirmed that the vesicles derived from bacteria were absorbed within 5 minutes after the administration, were systemically distributed, and were excreted through the kidney, liver and the like (see Example 1).

本発明の他の実施例では、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病などの患者にと年齢と性別が対応した正常人の大便、血液、小便又は唾液から分離した小胞を用いて細菌メタゲノム分析を実施した結果、正常人のサンプルに比べて胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病などの患者のサンプルでクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（実施例３〜実施例２０参照）。   In another embodiment of the present invention, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, atypical angina, chronic. Bacterial metagenomics using vesicles isolated from stool, blood, urine or saliva of normal subjects of age and sex, which correspond to patients with obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression As a result of the analysis, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, and dysmorphic narrowing compared to normal human samples. It was confirmed that vesicles derived from cupriawidus bacteria were significantly reduced in samples of patients with heart disease, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, and depression. (See Examples 3 to 20).

本発明のまた他の実施例では、前記実施例の結果を土台に、クプリアウィドゥス属細菌に属するクプリアウィドゥス・メタリデュランス種の細菌由来の小胞の特性を分析するために一層研究した結果、クプリアウィドゥス・メタリデュランス菌株を培養してそれから分泌された小胞が抗炎症効果を示すかを評価し、多様な濃度のクプリアウィドゥス・メタリデュランス由来の小胞を大食細胞に処理した後、炎症疾患の原因因子である大腸菌由来の小胞を処理して炎症媒介体の分泌を評価した結果、大腸菌由来の小胞によるＩＬ−６及びＴＮＦ−αの分泌をクプリアウィドゥス・メタリデュランス由来の小胞が効率的に抑制することを確認した（実施例２２参照）。   In still another embodiment of the present invention, based on the results of the above-mentioned embodiment, further studies were conducted to analyze the characteristics of vesicles derived from the bacteria of the genus Cupriavidus metallidurans belonging to the genus Cupriavidus. As a result, it was evaluated whether the vesicles secreted from the cupriawidus metallidurans strains were cultured and showed an anti-inflammatory effect, and vesicles derived from cupriawidus metallidurans at various concentrations were eaten fast. After treating the cells, the vesicles derived from Escherichia coli, which is a causative factor of inflammatory diseases, were treated to evaluate the secretion of inflammatory mediators. As a result, the secretion of IL-6 and TNF-α by the vesicles derived from Escherichia coli was It was confirmed that the vesicles derived from Widus metallidurans suppressed efficiently (see Example 22).

本発明のまた他の実施例では、クプリアウィドゥス・メタリデュランス菌株を培養してそれから分泌された小胞が抗癌治療効果を示すかを評価した。そのために、癌細胞株をマウスの皮下に注射して癌モデルを作り、クプリアウィドゥス・メタリデュランス由来の小胞を癌細胞株の処理４日前からマウスに経口又は腹腔で投与した後、２０日間癌組織のサイズを測定した結果、前記小胞を腹腔及び経口で投与した場合に、対照群に比べて癌組織のサイズが減少した。特に、経口投与した場合に顕著に減少していることを確認した（実施例２３参照）。   In yet another embodiment of the present invention, a cupriawidus metallidurans strain was cultured to evaluate whether vesicles secreted therefrom exhibited an anticancer therapeutic effect. Therefore, a cancer model was prepared by subcutaneously injecting a cancer cell line into a mouse, and vesicles derived from cupriawidus metallidurans were orally or intraperitoneally administered to the mouse from 4 days before the treatment of the cancer cell line. As a result of measuring the size of the cancer tissue for a day, the size of the cancer tissue was decreased when the vesicles were intraperitoneally or orally administered, as compared with the control group. In particular, it was confirmed that the amount was remarkably decreased when orally administered (see Example 23).

本発明による薬学的組成物は、薬学的に許容可能な担体を含んでいてもよい。前記薬学的に許容可能な担体は、製剤時に通常的に用いられるものであって、食塩水、滅菌水、リンゲル液、緩衝食塩水、シクロデキストリン、デキストロース溶液、マルトデキストリン溶液、グリセロール、エタノール、リボソームなどを含むが、これに限定されず、必要に応じて、抗酸化剤、緩衝液など他の通常の添加剤をさらに含むことができる。また、希釈剤、分散剤、界面活性剤、結合剤、潤滑剤などを付加的に添加して水溶液、懸濁液、乳濁液などのような注射用剤型、丸薬、カプセル、顆粒又は錠剤で製剤化することができる。適合な薬学的に許容される担体及び製剤化についてはレミントンの文献に開示されている方法を用いて各成分によって好ましく製剤化することができる。本発明の薬学的組成物は、剤型に特別な制限はないが、注射剤、吸入剤、皮膚外用剤又は経口摂取剤などに製剤化することができる。   The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one that is commonly used in the preparation, and includes saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, ribosome, etc. However, the present invention is not limited thereto, and may further include other usual additives such as an antioxidant and a buffer, if necessary. Further, diluents, dispersants, surfactants, binders, lubricants and the like are additionally added to prepare injectable forms such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets. Can be formulated in. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated with each component using the methods disclosed in Remington's literature. The dosage form of the pharmaceutical composition of the present invention is not particularly limited, but it can be formulated into an injection, an inhalant, an external preparation for the skin, an ingestion agent, and the like.

本発明の薬学的組成物は、目的とする方法によって経口投与するか非経口投与（例えば、静脈内、皮下、皮膚、鼻腔、気道に適用）でき、投与量は、患者の状態及び体重、疾病の程度、薬物形態、投与経路及び時間によって異なるが、当業者により適切に選択され得る。   The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, dermally, nasally, or applied to the respiratory tract) according to the intended method. Can be appropriately selected by those skilled in the art, though it depends on the degree, the drug form, the administration route and the time.

本発明による薬学的組成物は、薬学的に有効な量で投与する。本発明において、薬学的に有効な量は、医学的治療に適用可能な合理的なベネフィット／リスクの割合で疾患を治療するに十分な量を意味し、有効容量のレベルは、患者の疾患の種類、重症度、薬物の活性、薬物に対する敏感度、投与時間、投与経路及び排出の割合、治療期間、同時に用いられる薬物を含んだ要素及びその他医学分野によく知られた要素によって決定され得る。本発明による組成物は、個別治療剤で投与するか他の治療剤と併用して投与され得、従来の治療剤とは順次又は同時に投与され得、単一又は多重投与され得る。上記した要素を全て考慮して副作用なしに最小限の量で最大の効果を得ることができる量を投与することが重要であり、これは、当業者によって容易に決定され得る。   The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the level of effective dose is the level of the patient's disease. It can be determined by type, severity, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including drugs used at the same time and other factors well known in the medical field. The compositions according to the invention may be administered in individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents and may be administered in single or multiple doses. It is important to consider all the above mentioned factors and to administer an amount that can achieve the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

具体的に、本発明による薬学的組成物の有効量は、患者の年齢、性別、体重によって変わることができ、一般的には、体重１ｋｇ当たり０．００１〜１５０ｍｇ、好ましくは、０．０１〜１００ｍｇを毎日又は隔日投与するか、１日１回〜３回に分けて投与することができる。しかし、投与経路、肥満の重症度、性別、体重、年齢などによって増減できるので、前記投与量がいかなる方法でも本発明の範囲を限定するものではない。   Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary according to the age, sex, and body weight of the patient, generally 0.001 to 150 mg, preferably 0.01 to 150 mg per kg body weight. 100 mg can be administered daily or every other day, or once to three times a day in divided doses. However, since the dose can be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dose does not limit the scope of the present invention by any method.

本発明の吸入剤組成物は、有効成分を吸入剤にそのまま添加するか他の成分とともに用いてもよく、通常的な方法によって適切に用いてもよい。有効成分の混合量は、その使用目的（予防又は治療用）によって適切に決定され得る。   In the inhalant composition of the present invention, the active ingredient may be added to the inhalant as it is, or may be used together with other ingredients, and may be appropriately used by a usual method. The amount of the active ingredient mixed can be appropriately determined depending on the purpose of use (prophylactic or therapeutic).

本発明の食品組成物は、健康機能食品組成物を含む。本発明による食品組成物は、有効成分を食品にそのまま添加するか他の食品又は食品成分とともに用いてもよく、通常的な方法によって適切に用いてもよい。有効成分の混合量は、その使用目的（予防又は改善用）によって適切に決定され得る。一般的に、食品又は飲料の製造時に、本発明の組成物は、原料に対して１５重量％以下、好ましくは、１０重量％以下の量で添加される。しかし、健康及び衛生を目的とするか又は健康調節を目的とする長期間の摂取の場合には、前記量は前記範囲以下であってもよい。   The food composition of the present invention includes a health functional food composition. The food composition according to the present invention may be added to the food as it is, or may be used together with other foods or food ingredients, or may be appropriately used by a conventional method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (for prevention or improvement). Generally, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight, based on the raw materials, during the production of foods or beverages. However, for long-term ingestion for health and hygiene purposes or for health regulation, the amount may be below the range.

本発明の食品組成物は、指示された割合で必須成分として前記有効成分を含有すること以外に他の成分には特別な制限がなく、通常の飲料のように多様な香味剤又は天然炭水化物などを追加成分として含有することができる。上述した天然炭水化物の例は、モノサッカライド、例えば、ブドウ糖、果糖など；ジサッカライド、例えば、マルトース、スクロースなど；及びポリサッカライド、例えば、デキストリン、シクロデキストリンなどのような通常的な糖、及びキシリトール、ソルビトール、エリスリトールなどの糖アルコールである。上述したもの以外の香味剤として天然香味剤（ソーマチン、ステビア抽出物、例えば、レバウディオサイドＡ、グリシルヒジンなど）及び合成香味剤（サッカリン、アスパルテームなど）を好適に用いることができる。前記天然炭水化物の割合は、当業者の選択によって適切に決定され得る。   The food composition of the present invention has no particular limitation on other components other than containing the above-mentioned active ingredient as an essential ingredient in an indicated ratio, and various flavors or natural carbohydrates such as ordinary beverages. Can be included as an additional component. Examples of natural carbohydrates mentioned above are monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, conventional sugars such as dextrin, cyclodextrin and the like, and xylitol, Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract, for example, rebaudioside A, glycylhidine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be preferably used. The proportion of the natural carbohydrate can be appropriately determined by the selection of those skilled in the art.

上記外に、本発明の食品組成物は、様々な栄養剤、ビタミン、ミネラル（電解質）、合成風味剤及び天然風味剤などの風味剤、着色剤及び充填剤（チーズ、チョコレートなど）、ペクチン酸及びその塩、アルギン酸及びその塩、有機酸、保護性コロイド増粘剤、ｐＨ調節剤、安定化剤、防腐剤、グリセリン、アルコール、炭酸飲料に用いられる炭酸化剤などを含有することができる。このような成分は、独立的に又は組み合わせて用いることができる。このような添加剤の割合も当業者により適切に選択され得る。   In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid. And salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. Such components can be used independently or in combination. The proportion of such additives can also be appropriately selected by those skilled in the art.

以下、本発明の理解を助けるための好ましい実施例を提示する。しかし、下記の実施例は本発明をより容易に理解するために提供されるものに過ぎず、下記の実施例により本発明の内容が限定されるものではない。   Hereinafter, preferred embodiments will be presented to help understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

（試験例１．腸内細菌及び細菌由来の小胞の体内吸収、分布及び排泄様相の分析）
腸内細菌と細菌由来の小胞が胃腸管を通じて全身的に吸収されるかを評価するために次のような方法で実験を行った。マウスの胃腸に蛍光で標識した腸内細菌と腸内細菌由来の小胞をそれぞれ５０μｇの容量で胃腸管に投与し、０分、５分、３時間、６時間、１２時間後に蛍光を測定した。マウスの全体イメージを観察した結果、図１ａに示したように、細菌の場合には全身的に吸収されなかったが、細菌由来の小胞の場合には、投与５分後に全身的に吸収され、投与３時間後には、膀胱で蛍光が濃く観察されて、小胞が泌尿器系に排泄されることが分かった。小胞は、投与１２時間まで体内に存在することが分かった。 (Test Example 1. Analysis of in vivo absorption, distribution and excretion of intestinal bacteria and vesicles derived from bacteria)
In order to evaluate whether enterobacteria and vesicles derived from bacteria are systemically absorbed through the gastrointestinal tract, the following experiments were conducted. Fluorescently labeled enteric bacteria and vesicles derived from enteric bacteria were administered to the gastrointestinal tract of mice at a dose of 50 μg each, and the fluorescence was measured 0 minutes, 5 minutes, 3 hours, 6 hours, and 12 hours later. . As a result of observing the whole image of the mouse, as shown in Fig. 1a, in the case of bacteria, it was not systemically absorbed, but in the case of vesicles derived from bacteria, it was systemically absorbed 5 minutes after administration. After 3 hours from the administration, strong fluorescence was observed in the bladder, and it was found that the vesicles were excreted in the urinary system. Vesicles were found to be present in the body up to 12 hours after administration.

また、腸内細菌と腸内細菌由来の小胞が全身的に吸収された後、多くの臓器に浸潤された様相を評価するために、蛍光で標識した５０μｇの細菌と細菌由来の小胞を上記の方法のように投与した後、投与１２時間後に血液、心臓、肺、肝臓、腎臓、脾臓、脂肪、筋肉を採取した。採取した組職で蛍光を観察した結果、図１ｂに示したように、細菌由来の小胞が血液、心臓、肺、肝臓、脾臓、脂肪、筋肉、腎臓に分布したが、細菌は吸収されないことが分かった。   In addition, in order to evaluate the appearance of the intestinal bacteria and the vesicles derived from the intestinal bacteria being systemically absorbed and then infiltrated into many organs, 50 μg of fluorescently labeled bacteria and the vesicles derived from the bacteria were evaluated. After administration as described above, 12 hours after administration, blood, heart, lung, liver, kidney, spleen, fat and muscle were collected. As a result of observing fluorescence in the collected tissue, bacterial-derived vesicles were distributed in blood, heart, lung, liver, spleen, fat, muscle and kidney as shown in Fig. 1b, but bacteria were not absorbed. I understood.

（試験例２．臨床サンプルで細菌由来の小胞のメタゲノム分析）
血液、小便、大便又は唾液などの臨床サンプルを先に１０ｍｌチューブに入れ、遠心分離法（３，５００×ｇ、１０ｍｉｎ、４℃）で浮遊物を沈めて上澄液のみを新しい１０ｍｌチューブに移した。０．２２μｍのフィルタを用いて細菌及び異物を除去した後、遠心濾過機（ｃｅｎｔｒｉｆｕｇａｌ ｆｉｌｔｅｒｓ ５０ｋＤ）に移し、１５００×ｇ、４℃で１５分間遠心分離して５０ｋＤより小さい物質は捨てて１０ｍｌまで濃縮した。もう一度、０．２２μｍのフィルタ（ｆｉｌｔｅｒ）を用いてバクテリア及び異物を除去した後、Ｔｙｐｅ９０ｔｉローターで１５０，０００×ｇ、４℃で３時間の間超高速遠心分離方法を用いて上澄液を捨て、固まったペレット（ｐｅｌｌｅｔ）を生理食塩水（ＰＢＳ）でとかした。 (Test Example 2. Metagenomic analysis of bacterial vesicles in clinical samples)
Put a clinical sample such as blood, urine, stool or saliva into a 10 ml tube first, submerge the suspension by centrifugation (3,500 xg, 10 min, 4 ° C) and transfer only the supernatant to a new 10 ml tube. did. After removing bacteria and foreign substances using a 0.22 μm filter, transfer to a centrifugal filter (centrifugal filters 50 kD), centrifuge at 1500 × g at 4 ° C. for 15 minutes, discard substances smaller than 50 kD and concentrate to 10 ml. did. Once again, bacteria and foreign substances were removed using a 0.22 μm filter, and the supernatant was discarded using a Type 90ti rotor at 150,000 × g and 4 ° C. for 3 hours using an ultra-high speed centrifugation method. The solidified pellet was dissolved with physiological saline (PBS).

上記方法で分離した小胞１００μｌを１００℃でボイルして内部のＤＮＡを脂質の外から出るようにし、その後、氷に５分間冷やした。その後、残った浮遊物を除去するために、１０，０００×ｇ、４℃で３０分間遠心分離して上澄液のみを集めた。そして、Ｎａｎｏｄｒｏｐを用いてＤＮＡ量を定量した。以後、前記抽出されたＤＮＡに細菌由来のＤＮＡが存在するかを確認するために、下記表１に示した１６ｓ ｒＤＮＡプライマー（ｐｒｉｍｅｒ）でＰＣＲを行って、前記抽出された遺伝子に細菌由来の遺伝子が存在することを確認した。   100 μl of the vesicles separated by the above method was boiled at 100 ° C. to allow the internal DNA to come out of the lipid, and then cooled on ice for 5 minutes. Then, in order to remove the remaining suspended matter, centrifugation was performed at 10,000 × g at 4 ° C. for 30 minutes, and only the supernatant was collected. Then, the amount of DNA was quantified using Nanodrop. Thereafter, in order to confirm whether or not the bacterial DNA was present in the extracted DNA, PCR was performed with the 16s rDNA primer (primer) shown in Table 1 below, and the bacterial gene was extracted from the extracted gene. Was confirmed to exist.

上記方法で抽出したＤＮＡを上記の１６Ｓ ｒＤＮＡプライマーを用いて増幅した後にシークエンシングを行い（Ｉｌｌｕｍｉｎａ ＭｉＳｅｑ ｓｅｑｕｅｎｃｅｒ）、その結果をＳｔａｎｄａｒｄ Ｆｌｏｗｇｒａｍ Ｆｏｒｍａｔ（ＳＦＦ）ファイルに出力し、ＧＳ ＦＬＸ ｓｏｆｔｗａｒｅ（ｖ２．９）を用いてＳＦＦファイルをｓｅｑｕｅｎｃｅファイル（．ｆａｓｔａ）とｎｕｃｌｅｏｔｉｄｅ ｑｕａｌｉｔｙｓｃｏｒｅファイルに変換した後、リードの信用度評価を確認し、ｗｉｎｄｏｗ（２０ｂｐｓ）平均ｂａｓｅ ｃａｌｌ ａｃｃｕｒａｃｙが９９％未満（Ｐｈｒｅｄ ｓｃｏｒｅ＜２０）である部分を除去した。Ｏｐｅｒａｔｉｏｎａｌ Ｔａｘｏｎｏｍｙ Ｕｎｉｔ（ＯＴＵ）分析のためには、ＵＣＬＵＳＴとＵＳＥＡＲＣＨを用いてシーケンス類似度によってクラスタリングを行い、属（ｇｅｎｕｓ）は９４％、科（ｆａｍｉｌｙ）は９０％、目（ｏｒｄｅｒ）は８５％、網（ｃｌａｓｓ）は８０％、門（ｐｈｙｌｕｍ）は７５％のシーケンス類似度を基準にクラスタリングし、各ＯＴＵの門（ｐｈｙｌｕｍ）、網（ｃｌａｓｓ）、目（ｏｒｄｅｒ）、科（ｆａｍｉｌｙ）、属（ｇｅｎｕｓ）レベルの分類を行い、ＢＬＡＳＴＮとＧｒｅｅｎＧｅｎｅｓの１６Ｓ ＲＮＡシーケンスデータベース（１０８，４５３シーケンス）を用いて属レベルで９７％以上のシーケンス類似度を有する細菌をプロファイリングした（ＱＩＩＭＥ）。   The DNA extracted by the above method was amplified using the above 16S rDNA primer and then sequenced (Illumina MiSeq sequencer), and the result was output to a Standard Flowgram Format (SFF) file, and GS FLX software (v2.9). After converting the SFF file into a sequence file (.fasta) and a nucleotidetide quality scores file, the credit evaluation of the read is confirmed, and the window (20 bps) average base call accuracy is less than 99% (Phred score <20). Was removed. For Operational Taxonomy Unit (OTU) analysis, clustering is performed by sequence similarity using UCLUST and USARCH, 94% for genus, 90% for family, 85% for order, Clustering is performed based on a sequence similarity of 80% for the class and 75% for the phylum, and the phylum, net, order, family, family, and genus of each OTU are clustered. Genus) level classification was performed, and BLASTN and GreenGenes 16S RNA sequence databases (108,453 sequences) were used to profile bacteria having a sequence similarity of 97% or more at the genus level (QIIME).

（試験例３．胃癌患者の大便、血液及び小便細菌由来の小胞のメタゲノム分析）
試験例２の方法で胃癌患者６３人の大便と、性別と年齢をマッチングした正常対照群１２６人の大便を対象として、大便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の大便に比べて胃癌患者の大便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２及び図２ａ参照）。 (Test Example 3. Metagenomic analysis of vesicles derived from stool, blood and urine bacteria of gastric cancer patients)
After performing the metagenomic analysis by extracting the gene from the vesicles present in the stool, targeting the stool of 63 gastric cancer patients and the stool of 126 normal control groups matched with sex and age by the method of Test Example 2 , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly decreased in the stools of gastric cancer patients as compared with the stools of normal persons (see Table 2 and FIG. 2a).

試験例２の方法で胃癌患者６７人の血液と、性別と年齢が対応した正常対照群１９８人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて胃癌患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表３及び図２ｂ参照）。   After performing the metagenomic analysis by extracting the gene from the vesicles present in the blood of the blood of 67 gastric cancer patients and the blood of 198 normal control groups whose sex and age were matched by the method of Test Example 2 , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of gastric cancer patients compared with the blood of normal humans (see Table 3 and FIG. 2b).

また、試験例２の方法で胃癌患者６１人の小便と、性別と年齢が対応した正常対照群１２０人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて胃癌患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表４及び図２ｃ参照）。 In addition, for the urine of 61 patients with gastric cancer and the urine of 120 normal control groups of gender and age according to the method of Test Example 2, genes were extracted from the vesicles present in the urine for metagenomic analysis. Then, the distribution of vesicles derived from cupria widus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the urine of the gastric cancer patient as compared with the urine of the normal person (see Table 4 and FIG. 2c).

（試験例４．大腸癌患者の大便及び小便細菌由来の小胞のメタゲノム分析）
試験例２の方法で大腸癌患者５２人の大便と、性別と年齢が対応した正常対照群８３人の大便を対象として、大便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の大便に比べて大腸癌患者の大便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表５及び図３ａ参照）。 (Test Example 4. Metagenomic analysis of vesicles derived from stool and urine bacteria of patients with colorectal cancer)
By the method of Test Example 2, the stools of 52 colorectal cancer patients and the stools of 83 normal control groups corresponding to sex and age were subjected to metagenomic analysis by extracting genes from vesicles present in the stool. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the stools of patients with colorectal cancer compared to the stools of normal subjects (see Table 5 and FIG. 3a).

また、試験例２の方法で大腸癌患者３８人の小便と、性別と年齢が対応した正常対照群３８人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて大腸癌患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表６及び図３ｂ参照）。   In addition, for the urine of 38 colorectal cancer patients and the urine of 38 normal control groups of gender and age according to the method of Test Example 2, a gene was extracted from the vesicles present in the urine for metagenomic analysis. After that, the distribution of vesicles derived from Cupriawidus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the urine of the patients with colorectal cancer compared with the urine of the normal human (see Table 6 and FIG. 3b).

（試験例５．膵臓癌患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法で膵臓癌患者２９１人の血液と、性別と年齢が対応した正常対照群２９１人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて膵臓癌患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表７及び図４参照）。 (Test Example 5. Metagenome analysis of vesicles derived from blood bacteria of pancreatic cancer patient)
By the method of Test Example 2, the blood of 291 pancreatic cancer patients and the blood of 291 normal control groups of which sex and age were matched were subjected to metagenomic analysis by extracting genes from vesicles present in the blood. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in blood of pancreatic cancer patients as compared with blood of normal humans (see Table 7 and FIG. 4).

（試験例６．胆管癌患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法で胆管癌患者７９人の血液と、性別と年齢が対応したした正常対照群２５９人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて胆管癌患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表８及び図５参照）。 (Test Example 6. Metagenomic analysis of vesicles derived from blood bacteria of bile duct cancer patients)
The method of Test Example 2 was applied to the blood of 79 cholangiocarcinoma patients and the blood of 259 normal control groups of which sex and age were matched, and genes were extracted from the vesicles present in the blood for metagenome analysis. Then, the distribution of vesicles derived from cupria widus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of bile duct cancer patients as compared with the blood of normal humans (see Table 8 and FIG. 5).

（試験例７．乳癌患者の血液及び小便細菌由来の小胞のメタゲノム分析）
試験例２の方法で乳癌患者９６人の血液と、性別と年齢が対応したした正常対照群１９２人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて乳癌患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表９及び図６ａ参照）。 (Test Example 7. Metagenomic analysis of vesicles derived from blood and urine bacteria of breast cancer patients)
For the blood of 96 breast cancer patients and the blood of 192 normal control groups whose sex and age were matched by the method of Test Example 2, genes were extracted from vesicles present in the blood and metagenome analysis was performed. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of breast cancer patients compared with the blood of normal humans (see Table 9 and FIG. 6a).

試験例２の方法で乳癌患者１２７人の小便と、性別と年齢が対応した正常対照群２２０人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて乳癌患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１０及び図６ｂ参照）。   In the urine of 127 breast cancer patients and 220 urine of a normal control group of gender and age matched by the method of Test Example 2, genes were extracted from vesicles present in the urine and metagenome analysis was performed. , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly decreased in the urine of the breast cancer patient as compared with the urine of the normal person (see Table 10 and FIG. 6b).

（試験例８．卵巣癌患者の血液及び小便細菌由来の小胞のメタゲノム分析）
試験例２の方法で卵巣癌患者１３６人の血液と、性別と年齢が対応した正常対照群１３６人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて卵巣癌患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１１及び図７ａ参照）。 (Test Example 8. Metagenomic analysis of blood and urinary vesicle-derived vesicles of ovarian cancer patients)
By the method of Test Example 2, the blood of 136 ovarian cancer patients and the blood of 136 normal control groups corresponding to gender and age were subjected to metagenomic analysis by extracting genes from vesicles present in the blood. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of ovarian cancer patients compared with the blood of normal humans (see Table 11 and FIG. 7a).

実施例２の方法で卵巣癌患者１３６人の小便と、性別と年齢が対応した正常対照群１３６人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて卵巣癌患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１２及び図７ｂ参照）。   Targeting the urine of 136 ovarian cancer patients and the urine of the normal control group 136 of which sex and age were matched by the method of Example 2, genes were extracted from the vesicles present in the urine and metagenomic analysis was performed. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the urine of the ovarian cancer patient as compared with the urine of the normal person (see Table 12 and FIG. 7b).

（試験例９．膀胱癌患者の血液及び小便細菌由来の小胞のメタゲノム分析）
試験例２の方法で膀胱癌患者９６人の血液と、性別と年齢が対応した正常対照群１８４人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて膀胱癌患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１３及び図８ａ参照）。 (Test Example 9. Metagenomic analysis of blood and bladder cancer patient-derived vesicle-derived vesicles)
By the method of Test Example 2, the blood of 96 bladder cancer patients and the blood of 184 normal control groups corresponding to sex and age were subjected to gene extraction from vesicles present in the blood and metagenomic analysis was performed. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly decreased in the blood of the bladder cancer patient as compared with the blood of a normal person (see Table 13 and FIG. 8a).

また、試験例２の方法で膀胱癌患者９５人の小便と、性別と年齢が対応した正常対照群１５７人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて膀胱癌患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１４及び図８ｂ参照）。   In addition, for the urine of 95 bladder cancer patients and urine of the normal control group of 157 patients of the same sex and age by the method of Test Example 2, genes were extracted from the vesicles present in the urine for metagenomic analysis. After that, the distribution of vesicles derived from Cupriawidus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the urine of the bladder cancer patient as compared with the urine of the normal person (see Table 14 and FIG. 8b).

（試験例１０．前立腺癌患者の小便細菌由来の小胞のメタゲノム分析）
試験例２の方法で前立腺癌患者５３人の小便と、性別と年齢が対応した正常対照群１５９人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて前立腺癌患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１５及び図９参照）。 (Test Example 10. Metagenomic analysis of vesicles derived from urine bacteria of prostate cancer patients)
By the method of Test Example 2, targeting urine of 53 patients with prostate cancer and urine of 159 normal control group having matched sex and age, genes were extracted from vesicles present in the urine and metagenomic analysis was performed. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the urine of the prostate cancer patient as compared with the urine of the normal person (see Table 15 and FIG. 9).

（試験例１１．頭頸部癌患者の唾液細菌由来の小胞のメタゲノム分析）
試験例２の方法で頭頸部癌患者５７人の唾液と、正常対照群２７７人の唾液を対象として、唾液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の唾液に比べて頭頸部癌患者の唾液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１６及び図１０参照）。 (Test Example 11. Metagenomic analysis of vesicles derived from salivary bacteria of head and neck cancer patients)
Targeting saliva of 57 head and neck cancer patients and saliva of 277 normal control groups by the method of Test Example 2, genes were extracted from vesicles present in saliva and metagenomic analysis was performed, and then cupriawi was used. The distribution of vesicles from the genus Duss was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriawidus were significantly reduced in the saliva of the head and neck cancer patients as compared with the saliva of the normal person (see Table 16 and FIG. 10).

（試験例１２．リンパ腫患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法でリンパ腫患者５７人の血液と、性別と年齢をマッチングした正常対照群１６３人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べてリンパ腫患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１７及び図１１参照）。 (Test Example 12. Metagenomic analysis of vesicles derived from blood bacteria of lymphoma patient)
After performing the metagenomic analysis by extracting genes from vesicles present in the blood of 57 lymphoma patients' blood by the method of Test Example 2 and blood of 163 normal control groups whose sex and age were matched. , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of lymphoma patients as compared with the blood of normal persons (see Table 17 and FIG. 11).

（試験例１３．心臓疾患患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法で心筋病症患者７２人の血液と、性別と年齢が対応した正常対照群１６３人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて心筋病症患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１８及び図１２ａ参照）。 (Test Example 13. Metagenomic analysis of vesicles derived from blood bacteria of heart disease patient)
By the method of Test Example 2, the blood of 72 cardiomyopathy patients and the blood of 163 normal control groups corresponding to sex and age were subjected to metagenome analysis by extracting genes from vesicles present in the blood. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the blood of patients with cardiomyopathy compared to the blood of normal persons (see Table 18 and FIG. 12a).

実施例２の方法で心房細動患者３４人の血液と、性別と年齢が対応した正常対照群６３人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて心房細動患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表１９及び図１２ｂ参照）。   The method of Example 2 was applied to blood of 34 patients with atrial fibrillation and blood of 63 normal control groups corresponding to sex and age, and genes were extracted from vesicles present in the blood for metagenomic analysis. Then, the distribution of vesicles derived from cupria widus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the blood of patients with atrial fibrillation as compared with the blood of normal persons (see Table 19 and FIG. 12b).

試験例２の方法で異形狭心症患者８０人の血液と、性別と年齢が対応した正常対照群８０人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて異形狭心症患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２０及び図１２ｃ参照）。   Using the method of Test Example 2 for the blood of 80 patients with variant angina and the blood of 80 normal control groups of the same sex and age, genes were extracted from vesicles present in the blood for metagenomic analysis. After that, the distribution of vesicles derived from Cupriawidus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the blood of the patients with variant angina compared to the blood of normal persons (see Table 20 and FIG. 12c).

（試験例１４．脳卒中患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法で脳卒中患者１１５人の血液と、性別と年齢が対応した正常対照群１０９人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて脳卒中患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２１及び図１３参照）。 (Test Example 14. Metagenome analysis of vesicles derived from blood bacteria of stroke patient)
After performing the metagenomic analysis by extracting the gene from the vesicles present in the blood of the blood of 115 stroke patients and the blood of 109 normal control groups corresponding to sex and age by the method of Test Example 2 , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of stroke patients as compared with the blood of normal persons (see Table 21 and FIG. 13).

（試験例１５．慢性閉塞性肺疾患（ＣＯＰＤ）患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法でＣＯＰＤ患者２０５人の血液と、性別と年齢が対応した正常対照群２３１人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べてＣＯＰＤ患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２２及び図１４参照）。 (Test Example 15. Metagenomic analysis of vesicles derived from blood bacteria of patients with chronic obstructive pulmonary disease (COPD))
After performing the metagenomic analysis by extracting the gene from the vesicles present in the blood of the blood of 205 COPD patients and the blood of 231 normal control group whose sex and age were matched by the method of Test Example 2 , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of COPD patients compared with the blood of normal persons (see Table 22 and FIG. 14).

（試験例１６．糖尿病患者の血液、小便及び唾液細菌由来の小胞のメタゲノム分析）
試験例２の方法で糖尿病患者６１人の血液と、性別と年齢が対応した正常対照群１２２人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて糖尿病患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２３及び図１５ａ参照）。 (Test Example 16. Metagenomic analysis of vesicles derived from blood, urine and salivary bacteria of diabetic patients)
After the blood was extracted from the vesicles present in the blood of 61 diabetic patients and the blood of 122 normal control groups corresponding to sex and age by the method of Test Example 2 and metagenomic analysis was performed. , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of diabetic patients as compared with the blood of normal persons (see Table 23 and FIG. 15a).

実施例２の方法で糖尿病患者６０人の小便と、性別と年齢が対応した正常対照群１３４人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて糖尿病患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２４及び図１５ｂ参照）。   After targeting the urine of 60 diabetic patients and the urine of 134 normal controls grouped by sex and age by the method of Example 2, genes were extracted from vesicles present in the urine and metagenomic analysis was performed. , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the urine of diabetic patients as compared with the urine of normal persons (see Table 24 and FIG. 15b).

実施例２の方法で糖尿病患者３７人の唾液と、年齢が対応した正常対照群２７７人の唾液を対象として、唾液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の唾液に比べて糖尿病患者の唾液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２５及び図１５ｃ参照）。   Targeting saliva of 37 diabetic patients and 277 of age-matched normal control group by the method of Example 2, genes were extracted from vesicles present in saliva and metagenome analysis was performed. The distribution of vesicles derived from the genus Pryavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the saliva of diabetic patients as compared with the saliva of normal persons (see Table 25 and FIG. 15c).

（試験例１７．腎不全患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法で腎不全患者３２人の血液と、性別と年齢が対応した正常対照群３２人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて腎不全患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２６及び図１６参照）。 (Test Example 17. Metagenomic analysis of vesicles derived from blood bacteria of renal failure patient)
For the blood of 32 renal failure patients and the blood of 32 normal control groups corresponding to sex and age by the method of Test Example 2, genes were extracted from vesicles present in the blood and metagenomic analysis was performed. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that vesicles derived from the bacteria of the genus Cupriavidus were significantly reduced in the blood of patients with renal failure as compared with the blood of normal persons (see Table 26 and FIG. 16).

（試験例１８．認知症患者の血液細菌由来の小胞のメタゲノム分析）
試験例２の方法で認知症患者６７人の血液と、性別と年齢が対応した正常対照群７０人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の血液に比べて認知症患者の血液でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２７及び図１７参照）。 (Test Example 18. Metagenomic analysis of vesicles derived from blood bacteria of patients with dementia)
By the method of Test Example 2, blood was collected from 67 dementia patients and blood from 70 normal control groups corresponding to sex and age, and genes were extracted from vesicles present in the blood for metagenomic analysis. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the blood of patients with dementia as compared with the blood of normal persons (see Table 27 and FIG. 17).

（試験例１９．パーキンソン病患者の小便細菌由来の小胞のメタゲノム分析）
試験例２の方法でパーキンソン病患者３９人の小便と、性別と年齢が対応した正常対照群７６人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べてパーキンソン病患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２８及び図１８参照）。 (Test Example 19. Metagenomic analysis of vesicles derived from urine bacteria of patients with Parkinson's disease)
Genes were extracted from the vesicles present in the urine for the urine of 39 patients with Parkinson's disease and the urine of 76 normal control groups of the same sex and age by the method of Test Example 2 to perform metagenomic analysis. Then, the distribution of vesicles derived from the bacterium of the genus Cupriavidus was evaluated. As a result, it was confirmed that the urine derived from the bacteria of the genus Cupriawidus was significantly reduced in the urine of patients with Parkinson's disease as compared with the urine of normal persons (see Table 28 and FIG. 18).

（試験例２０．鬱病患者の小便細菌由来の小胞のメタゲノム分析）
試験例２の方法で鬱病患者２０人の小便と、性別と年齢をマッチングした正常対照群２１人の小便を対象として、小便内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、クプリアウィドゥス属細菌由来の小胞の分布を評価した。その結果、正常人の小便に比べて鬱病患者の小便でクプリアウィドゥス属細菌由来の小胞が有意に減少していることを確認した（表２９及び図１９参照）。 (Test Example 20. Metagenomic analysis of vesicles derived from urine bacteria of depressed patients)
After the urine of 20 patients with depression and the urine of 21 normal controls whose sex and age were matched by the method of Test Example 2 were subjected to metagenomic analysis by extracting genes from vesicles present in the urine. , And evaluated the distribution of vesicles derived from cupria widus bacteria. As a result, it was confirmed that the vesicles derived from the bacterium of the genus Cupriavidus were significantly reduced in the urine of the depressed patient as compared with the urine of the normal person (see Table 29 and FIG. 19).

（試験例２１．クプリアウィドゥス・メタリデュランス菌の培養及び小胞の分離）
クプリアウィドゥス・メタリデュランス（Ｃｕｐｒｉａｖｉｄｕｓ ｍｅｔａｌｌｉｄｕｒａｎｓ）菌株を培養した後、その小胞を分離して特性を分析した。クプリアウィドゥス・メタリデュランス菌株を２８℃の好気性チャンバで吸光度（ＯＤ６００）が１．０〜１．５になるまでＴＳＢ（Ｔｒｙｐｔｉｃ ｓｏｙ ｂｒｏｔｈ）培地で培養した後、ＬＢ（Ｌｕｒｉａ Ｂｅｒｔａｎｉ ｂｒｏｔｈ）培地に継代培養（ｓｕｂ−ｃｕｌｔｕｒｅ）した。以後、菌株が含まれている培地の上澄液を回収して１０，０００ｇ、４℃で２０分間遠心分離した後、菌株を除去して０．２２μｍのフィルタで濾過した。濾過した上澄液を１００ｋＤａ Ｐｅｌｌｉｃｏｎ ２ Ｃａｓｓｅｔｔｅのフィルタメンブレイン（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒｅ、ＵＳ）でＭａｓｔｅｒＦｌｅｘ ｐｕｍｐｓｙｓｔｅｍ（Ｃｏｌｅ−Ｐａｒｍｅｒ、ＵＳ）を用いて限外濾過（ｍｉｃｒｏｆｉｌｔｒａｔｉｏｎ）を通じて５０ｍｌの体積で濃縮した。濃縮した上澄液をもう一度０．２２μｍのフィルタで濾過した。その後、ＢＣＡ（Ｂｉｃｉｎｃｈｏｎｉｎｉｃ ａｃｉｄ）ａｓｓａｙを用いてタンパク質を定量し、得られた小胞に対して下記実験を実施した。 (Test Example 21. Culture of Cupriavidus metallidurans and isolation of vesicles)
After culturing the Cupriavidus metallidurans strain, the vesicles were isolated and characterized. The cupriawidus metallidurans strain was cultivated in a TSB (Tryptic soy broth) medium in an aerobic chamber at 28 ° C. until the absorbance (OD 600 ) reached 1.0 to 1.5, and then LB (Luria Bertani broth). Subculture was performed on the medium. Thereafter, the supernatant of the medium containing the strain was collected and centrifuged at 10,000 g at 4 ° C. for 20 minutes, the strain was removed, and the mixture was filtered through a 0.22 μm filter. The filtered supernatant was concentrated on a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore, US) using a MasterFlex pumpsystem (Cole-Parmer, US) in a volume of 50 ml through ultrafiltration. The concentrated supernatant was filtered again with a 0.22 μm filter. After that, the protein was quantified using BCA (Bicinchonic acid) assay, and the following experiment was carried out on the obtained vesicles.

（試験例２２．クプリアウィドゥス・メタリデュランス由来の小胞の抗炎症効果）
炎症細胞でクプリアウィドゥス・メタリデュランス由来の小胞の細胞死滅に対する影響を確認するために、マウス大食細胞株であるＲａｗ ２６４．７細胞にクプリアウィドゥス・メタリデュランス由来の小胞（Ｃ．ｍｅｔａｌｌｉｄｕｒａｎｓ ＥＶ）を多様な濃度（０．１、１、１０μｇ／ｍｌ）で処理した後、細胞生存力測定（Ｃｅｌｌ ｖｉａｂｉｌｉｔｙ ｔｅｓｔ）を行った。より具体的に、４８−ｗｅｌｌの細胞培養プレート中に４×１０４個ずつ分注したＲａｗ ２６４．７細胞に、ＤＭＥＭ（Ｄｕｌｂｅｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ）無血清培地を入れた多様な濃度のクプリアウィドゥス・メタリデュランス由来の小胞を処理して１２時間の間培養した。その後、細胞にＥＺ−ＣＹＴＯＸ（Ｄｏｇｅｎ、Ｋｏｒｅａ）を４時間の間処理した後、ＳｐｅｃｔｒａＭａｘ Ｍ３ ｍｉｃｒｏｐｌａｔｅ ｒｅａｄｅｒ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｅｓ、ＵＳＡ）を用いて４５０ｎｍで吸光度を測定した。その結果、図２０に示したように、クプリアウィドゥス・メタリデュランス由来の小胞を大食細胞株に処理したとき、細胞死滅が誘導されないことを確認した。 (Test Example 22. Anti-inflammatory effect of vesicles derived from Cupriavidus metallidurans)
In order to confirm the effect on cell death of vesicles derived from Cupriavidus metallidurans in inflammatory cells, vesicles derived from Cupriavidus metallidurans were introduced into Raw 264.7 cells, which is a mouse macrophage cell line ( C. metallidurans EV) was treated with various concentrations (0.1, 1, 10 μg / ml), and then cell viability test (Cell viability test) was performed. More specifically, various concentrations were obtained by adding DMEM (Dulbeco's Modified Eagle's Medium) serum-free medium to Raw 264.7 cells dispensed in 4 × 10 4 cells in a 48-well cell culture plate. The vesicles derived from Cupriawidus metallidurans were treated and cultured for 12 hours. Thereafter, the cells were treated with EZ-CYTOX (Dogen, Korea) for 4 hours, and then the absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA). As a result, as shown in FIG. 20, it was confirmed that cell killing was not induced when the vesicles derived from Cupriavidus metallidurans were treated with a macrophage cell line.

上記結果を土台に、クプリアウィドゥス・メタリデュランス由来の小胞の抗炎症効果を評価するために、多様な濃度（０．１、１、１０μｇ／ｍｌ）のクプリアウィドゥス・メタリデュランス由来の小胞を大食細胞株に１２時間前処理した後、病原性小胞である大腸菌由来の小胞１μｇ／ｍｌを処理し、１２時間後に炎症性サイトカインの分泌をＥＬＩＳＡで測定した。ＥＬＩＳＡを行うために、キャプチャー（Ｃａｐｔｕｒｅ）抗体をリン酸緩衝生理食塩水（ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ、ＰＢＳ）に希釈して９６ ｗｅｌｌのポリスチレン（ｐｏｌｙｓｔｙｒｅｎｅ）プレートに作用濃度に合わせて５０μｌずつ分注した後、４℃で一晩の間反応させた。   Based on the above results, in order to evaluate the anti-inflammatory effect of cupriawidus metalliduran-derived vesicles, various concentrations (0.1, 1, 10 μg / ml) of cupriawidus metalliduran Vesicles were treated with macrophage cell lines for 12 hours, then treated with 1 μg / ml of vesicles derived from Escherichia coli, which is a pathogenic vesicle, and 12 hours later, inflammatory cytokine secretion was measured by ELISA. In order to carry out ELISA, a capture antibody is diluted in phosphate buffered saline (PBS) and dispensed into a 96-well polystyrene (polystyrene) plate according to the working concentration by 50 μl each. The reaction was allowed to proceed overnight at 4 ° C.

その後、ＰＢＳＴ（０．０５% ｔｗｅｅｎ−２０が入っているリン酸緩衝生理食塩水）溶液１００μｌで３回ずつ洗浄した後、ＲＤ（１％ ＢＳＡが入っているリン酸緩衝生理食塩水）溶液１００μｌを分注して常温で１時間の間ブロッキング（ｂｌｏｃｋｉｎｇ）し、サンプル及びスタンダード（ｓｔａｎｄａｒｄ）は、濃度に合わせて５０μｌず分注して常温で２時間の間反応させた。その後、ＰＢＳＴ １００μｌで３回洗浄した後、検出（ｄｅｔｅｃｔｉｏｎ）抗体をＲＤに希釈して作用濃度に合わせて５０μｌずつ分注して常温で２時間の間反応させ、ＰＢＳＴ １００μｌで３回洗浄した後、Ｓｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ（Ｒ＆Ｄ ｓｙｓｔｅｍ、ＵＳＡ）をＲＤに１／４０で希釈して５０μｌずつ分注して常温で２０分間反応させた。   Then, after washing three times with 100 μl of PBST (phosphate buffered saline containing 0.05% tween-20) each, 100 μl of RD (phosphate buffered saline containing 1% BSA) solution Was dispensed and blocked at room temperature for 1 hour, and 50 μl of the sample and standard were dispensed according to the concentration and reacted at room temperature for 2 hours. Then, after washing with 100 μl of PBST three times, the detection antibody was diluted in RD and dispensed in 50 μl aliquots according to the working concentration, reacted at room temperature for 2 hours, and washed with 100 μl of PBST three times. , Streptavidin-HRP (R & D system, USA) was diluted to 1/40 in RD, dispensed in 50 μl aliquots, and reacted at room temperature for 20 minutes.

最後に、ＰＢＳＴ １００μｌで３回洗浄した後、ＴＭＢ基質（ＳｕｒＭｏｄｉｃｓ、ＵＳＡ）５０μｌを分注して５分〜２０分後に発色が進行した時点で、１Ｍの硫酸溶液を５０μｌずつ分注して反応を止め、ＳｐｅｃｔｒａＭａｘ Ｍ３ ｍｉｃｒｏｐｌａｔｅ ｒｅａｄｅｒ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ、ＵＳＡ）を用いて４５０ｎｍで吸光度を測定した。   Finally, after washing 3 times with 100 μl of PBST, 50 μl of TMB substrate (SurModics, USA) was dispensed, and 5 to 20 minutes later, when color development proceeded, 50 μl of 1 M sulfuric acid solution was dispensed and reacted. Then, the absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA).

その結果、図２１に示したように、クプリアウィドゥス・メタリデュランス由来の小胞を前処理した場合、大腸菌由来の小胞によるＩＬ−６及びＴＮＦ−αの分泌が顕著に抑制されることを確認した。特に、クプリアウィドゥス・メタリデュランス由来の小胞の前処理によるＴＮＦ−αの分泌抑制効果が有用微生物対照群であるラクトバシラス・プランタルム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）由来の小胞の前処理によるＴＮＦ−αの分泌抑制効果より著しく大きいことを確認した。上記結果は、大腸菌由来の小胞のような病原性小胞により誘導される炎症反応をクプリアウィドゥス・メタリデュランス由来の小胞が効率的に抑制できることを意味する。   As a result, as shown in FIG. 21, when pretreatment of cupriawidus metalliduran-derived vesicles, secretion of IL-6 and TNF-α by E. coli-derived vesicles was remarkably suppressed. It was confirmed. In particular, the effect of suppressing the secretion of TNF-α by pretreatment of vesicles derived from Cupriawidus metallidurans is useful. A microbial control group of Lactobacillus plantarum (Lactobacillus plantarum) It was confirmed that it was significantly larger than the secretion suppressing effect. The above results mean that the vesicles derived from Cupriavidus metalidurans can efficiently suppress the inflammatory response induced by pathogenic vesicles such as Escherichia coli-derived vesicles.

（試験例２３．クプリアウィドゥス・メタリデュランス由来の小胞の抗癌効果）
前記試験例を土台に、クプリアウィドゥス・メタリデュランス由来の小胞の抗癌効果を確認した。それのために、図２２に示したように、クプリアウィドゥス・メタリデュランス菌株（ＣＭＴ１０１）由来の小胞を６週齢のＣ５７ＢＬ／６の雄マウスに腹腔注射又は経口で投与し、投与４日目に癌細胞株（ＣＴ２６ ｃｅｌｌ）を皮下に注射して癌モデルを作った。癌細胞株を投与した後、クプリアウィドゥス・メタリデュランス菌株由来の小胞を腹腔注射又は経口で毎日投与して２４日目まで癌組織のサイズを測定した。その結果、図２３に示したように、癌組織のサイズは、対照群である生理食塩水経口投与群に比べて前記小胞を腹腔注射で投与したマウスと経口で投与したマウスで癌組織のサイズが減少した。特に、経口で投与した場合にサイズが一層減少した。上記結果は、クプリアウィドゥス・メタリデュランス由来の小胞を投与したとき、癌組織の成長を効率的に抑制することができることを意味する。 (Test Example 23. Anticancer effect of vesicles derived from Cupriavidus metallidurans)
Based on the above test example, the anticancer effect of the vesicles derived from Cupriavidus metallidulans was confirmed. For that purpose, as shown in FIG. 22, vesicles derived from the cupriawidus metallidurans strain (CMT101) were intraperitoneally injected or orally administered to 6-week-old C57BL / 6 male mice, and administration 4 On the day, a cancer cell line (CT26 cell) was subcutaneously injected to make a cancer model. After administration of the cancer cell line, vesicles derived from the Cupriawidus metallidurans strain were administered daily by intraperitoneal injection or oral administration, and the size of the cancer tissue was measured until the 24th day. As a result, as shown in FIG. 23, the size of the cancer tissue was larger in the mice treated with the vesicles by intraperitoneal injection and the mice administered with the oral cavity than in the control group of the saline orally administered group. Reduced size. In particular, the size was further reduced when administered orally. The above results mean that the growth of cancer tissue can be effectively suppressed when the vesicles derived from Cupriavidus metallidurans are administered.

上述した本発明の説明は例示のためのもので、本発明が属する技術分野において通常の知識を有した者は、本発明の技術的思想や必須的な特徴を変更しなくても他の具体的な形態に容易に変形が可能であることが理解できる。したがって、以上で記述した実施例は、全ての面で例示的なものであり、限定的ではないものと理解すべきである。   The above description of the present invention is provided for the purpose of illustration, and a person having ordinary knowledge in the technical field to which the present invention belongs does not need to change the technical idea or essential features of the present invention to obtain other specific examples. It can be understood that the shape can be easily modified. Therefore, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive.

本発明によるクプリアウィドゥス属細菌由来の小胞は、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病に対する診断方法だけでなく、前記疾患に対する予防、改善又は治療用組成物でも用いられるので、関連医療及び食品産業分野に有用に用いられ得ると期待される。   Vesicles derived from cupriawidus bacterium according to the present invention are gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, Variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, as well as a diagnostic method for Parkinson's disease or depression, since it is also used in a preventive, ameliorating or therapeutic composition for said disease, it is a related medical treatment. And is expected to be useful in the field of food industry.

Claims (14)

下記のステップを含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の診断のための情報を提供する方法：
（ａ）正常人及び被検者のサンプルから分離した細胞外小胞からＤＮＡを抽出するステップ；
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）を行った後、それぞれのＰＣＲ産物を収得するステップ；及び
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常人に比べてクプリアウィドゥス（Ｃｕｐｒｉａｖｉｄｕｓ）属細菌由来の細胞外小胞の含量が低い場合、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病に分類するステップ。 Gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease including the following steps To provide information for the diagnosis of stroke, diabetes, renal failure, dementia, Parkinson's disease or depression:
(A) extracting DNA from extracellular vesicles separated from normal and subject samples;
(B) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on a gene sequence present in 16S rDNA, and then collecting each PCR product; and (c) ) When the content of extracellular vesicles derived from a bacterium of the genus Cupriavidus is lower than that of a normal person through quantitative analysis of the PCR product, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, Bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression. 前記ステップ（ａ）でのサンプルは、大便、血液、小便又は唾液であることを特徴とする、請求項１に記載の情報提供方法。   The information providing method according to claim 1, wherein the sample in step (a) is stool, blood, urine, or saliva. クプリアウィドゥス（Ｃｕｐｒｉａｖｉｄｕｓ）属細菌由来の小胞を有効成分として含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病からなる群より選択される１以上の疾病の予防又は治療用である、薬学的組成物。   Gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atria containing vesicles derived from bacteria of the genus Cupriavidus as an active ingredient Pharmaceutical composition for the prevention or treatment of one or more diseases selected from the group consisting of fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression. object. 前記小胞は、平均直径が１０〜２００ｎｍであることを特徴とする、請求項３に記載の薬学的組成物。   The pharmaceutical composition according to claim 3, wherein the vesicles have an average diameter of 10 to 200 nm. 前記小胞は、クプリアウィドゥス属細菌から自然的又は人工的に分泌されることを特徴とする、請求項３に記載の薬学的組成物。   The pharmaceutical composition according to claim 3, wherein the vesicle is naturally or artificially secreted from a bacterium belonging to the genus Cupriavidus. 前記クプリアウィドゥス属細菌由来の小胞は、クプリアウィドゥス・メタリデュランス（Ｃｕｐｒｉａｖｉｄｕｓｍｅｔａｌｌｉｄｕｒａｎｓ）から分泌されることを特徴とする、請求項３に記載の薬学的組成物。   The pharmaceutical composition according to claim 3, wherein the vesicle derived from a bacterium of the genus Cupriavidus is secreted from Cupriavidus metallidurans. クプリアウィドゥス（Ｃｕｐｒｉａｖｉｄｕｓ）属細菌由来の小胞を有効成分として含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病からなる群より選択される１以上の疾病予防又は改善用食品組成物。   Gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atria containing vesicles derived from bacteria of the genus Cupriavidus as an active ingredient A food composition for preventing or improving one or more diseases selected from the group consisting of fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression. 前記小胞は、平均直径が１０〜２００ｎｍであることを特徴とする、請求項７に記載の食品組成物。   The food composition according to claim 7, wherein the vesicles have an average diameter of 10 to 200 nm. 前記小胞は、クプリアウィドゥス属細菌から自然的又は人工的に分泌されることを特徴とする、請求項７に記載の食品組成物。   The food composition according to claim 7, wherein the vesicles are naturally or artificially secreted from a bacterium belonging to the genus Cupriavidus. 前記クプリアウィドゥス属細菌由来の小胞は、クプリアウィドゥス・メタリデュランス（Ｃｕｐｒｉａｖｉｄｕｓｍｅｔａｌｌｉｄｕｒａｎｓ）から分泌されることを特徴とする、請求項７に記載の食品組成物。   The food composition according to claim 7, wherein the vesicles derived from the bacterium of the genus Cupriavidus are secreted from Cupriavidus metallidurans. クプリアウィドゥス（Ｃｕｐｒｉａｖｉｄｕｓ）属細菌由来の小胞を有効成分として含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病からなる群より選択される１以上の疾病の予防又は治療用である、吸入剤組成物。   Gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atria containing vesicles derived from bacteria of the genus Cupriavidus as an active ingredient Inhalant composition for preventing or treating one or more diseases selected from the group consisting of fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression object. 下記のステップを含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病の診断方法：
（ａ）正常人及び被検者のサンプルから分離した細胞外小胞からＤＮＡを抽出するステップ；
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲを行った後、それぞれのＰＣＲ産物を収得するステップ；及び
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常人に比べてクプリアウィドゥス（Ｃｕｐｒｉａｖｉｄｕｓ）属細菌由来の細胞外小胞の含量が低い場合、胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病又は鬱病に分類するステップ。 Gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina pectoris, chronic obstructive pulmonary disease including the following steps Methods for diagnosing stroke, diabetes, renal failure, dementia, Parkinson's disease or depression:
(A) extracting DNA from extracellular vesicles separated from normal and subject samples;
(B) performing PCR on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, and then obtaining each PCR product; and (c) the PCR product Gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer when the content of extracellular vesicles derived from cupriavidus bacteria is lower than that of normal people through quantitative analysis. , Head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression. クプリアウィドゥス属細菌由来の小胞を有効成分として含む薬学的組成物を個体に投与するステップを含む胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病からなる群より選択される１以上の疾病の予防又は治療方法。   Gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck comprising a step of administering to an individual a pharmaceutical composition containing vesicles derived from cupriawidus bacteria as an active ingredient Cancer, lymphoma, cardiomyopathy, atrial fibrillation, variant angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression Or a treatment method. クプリアウィドゥス属細菌由来の小胞の胃癌、大腸癌、膵臓癌、胆管癌、乳癌、卵巣癌、膀胱癌、前立腺癌、頭頸部癌、リンパ腫、心筋病症、心房細動、異形狭心症、慢性閉塞性肺疾患、脳卒中、糖尿病、腎不全、認知症、パーキンソン病及び鬱病からなる群より選択される１以上の疾病の予防又は治療のための使用。   Follicle derived from cupriawidus gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysmorphic angina Use for the prevention or treatment of one or more diseases selected from the group consisting of chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression.
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